Cell Bio Exam 2 Lecture 1

Front 1

Abasic sites, Deamination, Oxidative damage, Strand breaks, and Mismatches
are types of spontaneous/non-spontaneous mutations?

Back 1

Spontaneous

Front 2

Ionizing radiation
Ultraviolet radiation, Alkylating agents, and other chemical agents are examples of spontaneous/non-spontaneous mutations?

Back 2

Non-spontaneous

Front 3

In the deamination reaction, adenine is converted to what molecule?
This molecule is capable of H bonding to what base?

Back 3

Hypoxanthine.
Cytosine

Front 4

Give the product of the following deamination reactions:

Cytosine:
Adenine:

Back 4

Front 5

What molecule does 5-methyl-cytosine become when it is deaminated?

Back 5

Front 6

What are two consequences of deamination reactions?

Back 6

mispairing and base pair substitutions.

Front 7

Oxidative DNA Damage causes _________ and break down products including __________.
What is a common cause of oxidative DNA damage?

Back 7

ring saturation
Strand breaks.

Free radicals

Front 8

Two common products of oxidative DNA damage are?

Back 8

8-oxyguanine or thymine glycol

Front 9

Thymine undergoes oxidative damage and becomes what molecule?

Back 9

Front 10

What type of DNA strand break is the most lethal?

Back 10

Double strand break.

Front 11

Which spectrum of UV light is arguably environmentally irrelevant? Why?

Back 11

UV-C. B/c it is absorbed mostly by ozone.

Front 12

What form of UV light (and wavelength) is most commonly used in the lab?

Back 12

254 nm UV-C

Front 13

True/false: Only UV B and C produce oxidative damage?

Back 13

False! all three forms A, B, and C cause oxidative damage.

Front 14

What damage does UV do to DNA? Give the two types

Back 14

It results in the covalent linkage of two pyramidines. The most frequent type results in Cylobutane. The other common form results in 6-4 photoproducts

Front 15

6-4 photoproducts are common products of damage to DNA via UV exposure. What are these products so harmful?

Back 15

They create major helix distortion.

Front 16

Alkylation of guanine results in what molecule?

Back 16

O6-methylguanine

Front 17

What problem does alkylation cause?

Back 17

Interferes with base pairing

Front 18

Bifunctional alkylation results in _________. What is this detrimental to DNA?

Back 18

DNA Crosslinks.
This is a covalent crossbridge that can't be separated by DNA Helicase. Ergo, transcription and translation are messed up.

Front 19

What is the cancerous agent in tobacco?

Back 19

Benz[a]pyrene-guanine adduct.

Front 20

How do you study DNA repair genetically?

Back 20

Isolate mutants that are sensitive to DNA damage agent. Characterize their phenotype towards various agents. Then establish epistasis groups.

Front 21

What is photoreactivation? What is the experimental implication of this concept?

Back 21

Monomerization of pyrimidine dimers using a single enzyme and long-wave UV.


You must do UV experiments in the dark b/c light will cause your induced mutations to return to normal.

Front 22

What are the two DNA repair systems that work by direct reversal?

Back 22

Photoreactivation and alkyltransferase

Front 23

How many enzymes are required in the alkyl transferase repair mechanism?

What DNA problems does this repair mechanism fix and what molecules does it use?

What is special about this enzyme?

Back 23

One.

It can repair O6-Methylguanine and O4-Methyladenine. It uses O6-methylguanine methyl transferase or O4-methyladenine methyl transferase.

It is a suicide enzyme: it is used once and it is consumed.

Front 24

Give the mechanism for Base Excision Repair.

Back 24

Front 25

T/F: Base excision repair is an error free mechanism?

Back 25

True

Front 26

What are the different polymerases that can handle Base Excision Repair?

Back 26

Pol 2, 3, and DNA Pol Beta.

Front 27

What is the major differences between Base Excision Repair and Nucleotide Excision Repair?

1) # Nucleotides removed
2) Glycosylases/Enzyme specificity
3) Excision Site?

Back 27

1) Base Excision Repair removes only one base at a time. NER removes an oligo fragment.

2) Glycosylases of BER have a narrow substrate specificity. Enzymes of NER have wide substrate specificity and act on bulky damage that distorts double helix.

3)BER: Creation of an AP site and finally a 1 nt gap that is substrate to various modes of repair. NER: Bimodal incision creates a single-stranded gap that can be filled by repair synthesis

Front 28

Give the NER mechanism in Prokaryotes

Back 28

Front 29

Explain the concept of preferential repair.

Back 29

More important DNA is transcribed first. Exons and important genetic products are repaired before introns and non-coding sequences.

Also, works on the level of coding/non-coding strand. The transcribed DNA stand is repaired before the non-transcribed strand.

Front 30

In both pro and eukaryotes, what attracts the NER to a damaged strand?

Back 30

The RNA polymerase that is stuck at a lesion.

Front 31

Compare NER in E. Coli to eukaryotes (4).

-Basic Mechanism
-# of proteins involved, give example.
-# of helicases
-Type of incision and length excised oligonucleotide.

Back 31

Basic mechanism is the same, however there is no homology in the machinery.

More proteins are involved, including Transcription factor TFIIH

Eukaryotes have two helicases of different polarity that create the bubble.

As in E. coli, there is a bimodal coordinated incision but the excised oligonucleotide is of slightly different length

Front 32

Patients with Xeroderma Pigmentosum have a defect in what?

Back 32

NER

Front 33

Compare NER of preferential repair sites with global repair in Eukaryotes. Which one is CSA, B involved with and what syndrome is associated with it?

Back 33

Front 34

How does mismatch repair machinery know the new, wrongly inserted base from the template base?

Back 34

Methylation is the main strand discrimination mechanism! You have a small window where you can discriminate…this is when the machinery works (previous slide).

Front 35

Give the mechanism of Mismatch repair.

Back 35

MutS recognizes mismatch, MutH binds to hemimethylated sites (6-methyl adenine) for strand discrimination and cuts unmethylated strand
Incision and removal of a large DNA fragment

Front 36

What mechanism fixes slippage events and mismatched bases?

Back 36

Mismatch repair

Front 37

Repetitive regions are prone to __________. Give an example.

Back 37

insertion/deletion mismatches

SNPs

Front 38

T/F: Eukaryotes use Methylation for strand discrimination and have MutH homologs like prokaryotes.

Back 38

False! The mechanism is uknown.

Front 39

Cancer-prone syndrome with defect in mismatch repair and consequently microsatellite instability is?

Back 39

HNPCC (hereditary nonpolyposis colorectal cancer)

Front 40

T/F: Mismatch repair is a type of Nucleotide excision repair.

Back 40

False! It removes a fragment of oligonucleotides like mismatch repair, but otherwise it isn't the same.

Front 41

What is the ames test?

Back 41

A test for mutagenicity: place your mutants on an Ab treated plate. If they mutate back they can grow and this suggests your agent is too mutagenic for study.

Front 42

Compare transition to transversion.

Back 42

Pur--> Pur or Pyr--> Pyr is a transition.

Pur--> pyr is transversion

Front 43

What is a hypomorphic mutation?

Back 43

not complete inactivation of product

Front 44

T/F: Mutations due to direct mispairing during replication at sites of damage are rare

Back 44

True

Front 45

What is translesion synthesis? Give the mechanism.

Back 45

A tolerance mechanism that allows the completion of DNA synthesis in the presence of DNA damage

Front 46

How were translesion polymerases discovered?

Back 46

Discovery of non-mutable mutants proves the active nature of DNA damage-induced mutagenesis.

Front 47

What are the components of Pol V? What is the fxn of this polymerase?

Back 47

UmuC/UmuD. A bypass polymerase.

Front 48

What is SOS repair. How does it work?

Back 48

All of these genes are repressed the same way. Coordinated repression. These are all expressed during DNA damage…SOS repair!

Front 49

Genes that are required for damage-induced mutagenesis are induced by DNA damage. Why?

Back 49

Increases survival through damage tolerance.
Increases genetic variability in an adverse environment: inducible evolution (problem in environment stimulates better adapted cells to grow).

Front 50

True/False: Bypass polymerases have low processivity.

Back 50

True

Front 51

Give the general scheme for bypass polymerases

Back 51

Front 52

Give the following characteristics of Bypass polymerases:

Processivity
Exonuclease activity
Which ones are required for mutations?
Which one can bypass bypass cyclo-butane type pyrimidine dimers error-free? How does it do this?

Back 52

Low processivity

No 3'-->5' exonuclease activity (proofreading. Increases error rate)

Zeta, kappa, iota required for mutations

eta; Inserts AA opposite a TT dimer ---it looks for watson-crick BP instead of GEOMETRY like other pols.